JP6076962B2 - Heat-sensitive mucoadhesive or skin-adhesive penetration enhancers for local delivery of therapeutic agents - Google Patents

Description

  The present invention relates to a heat-sensitive mucoadhesive or skin-adhesive penetration enhancer for local delivery of therapeutic agents.

  This application claims the benefit of US Provisional Patent Application No. 61/470551 filed on April 1, 2011 and 61/602769 filed February 24, 2012, all figures The entire disclosure, including tables, and nucleic acid sequences, is hereby incorporated by reference.

  Cervical cancer is a life-threatening disease unless it is detected early and treated. 99.7% of cervical cancer cases are pathologically related to at least one of 15-18 carcinogenic human papillomaviruses (HPV) (see Non-Patent Documents 4 to 6). More than 85% of cervical cancers occur in other medically depopulated areas with historically few medical resources, where cancer is the leading cause of death in developing countries and women (see Non-Patent Document 1). Diagnosis and treatment at the stage of precancerous lesions or early cervical cancer can in most cases prevent progression to high-grade cervical cancer.

Garcia M, Jemal, A., Ward, E. M., Center, M. M., Hao, Y., Siegel, R. L., and Thun, M. J. 2008.Global Cancer Facts & Figures 2007. Atlanta, GA: American Cancer Society de Sanjose S et al., Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. Lancet Oncol 11: 1048-56 Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, Snijders PJ, Meijer CJ. 2003. Epidemiologic classification of human papillomavirus types associated with cervical cancer.N Engl J Med 348: 518-27 Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, Snijders PJ, Peto J, Meijer CJ, Munoz N. 1999. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 189: 12- 9 Hsu SI, Yang CM, Sim KG, Hentschel DM, O'Leary E, Bonventre JV. 2001. TRIP-Br: a novel family of PHD zinc finger- and bromodomain-interacting proteins that regulate the transcriptional activity of E2F-1 / DP -1. EMBO J 20: 2273-85 Sim KG, Zang Z, Yang CM, Bonventre JV, Hsu SI. 2004. TRIP-Br links E2F to novel functions in the regulation of cyclin E expression during cell cycle progression and in the maintenance of genomic stability.Cell Cycle 3: 1296- 304 Zang ZJ, Sim KG, Cheong JK, Yang CM, Yap CS, Hsu SI. 2007. Exploiting the TRIP-Br Family of Cell Cycle Regulatory Proteins as Chemotherapeutic Drug Targets in Human Cancer.Cancer Biol Ther 6 DeMay M. 2007. Practical principles of cytopathology (Revised edition) .Chicago: American Society for Clinical Pathology Press Stoughton RB. 1982. Enhanced percutaneous penetration with 1-dodecylazacycloheptan-2-one. Arch Dermatol 118: 474-7 Stoughton RB, and McClure, W. O. 1983. Azone; a new non-toxic enhancer of cutaneous penetration.Drug Development and Industrial Pharmacy 9: 725-44 Kaushik D, Batheja P, Kilfoyle B, Rai V, Michniak-Kohn B. 2008. Percutaneous permeation modifiers: enhancement versus retardation. Expert Opin Drug Deliv 5: 517-29 Kaushik D, Costache A, Michniak-Kohn B. 2010. Percutaneous penetration modifiers and formulation effects. Int J Pharm 386: 42-51 Kaushik D, Michniak-Kohn B. 2010. Percutaneous penetration modifiers and formulation effects: thermal and spectral analyzes. AAPS PharmSciTech 11: 1068-83 Spruance SL, McKeough M, Sugibayashi K, Robertson F, Gaede P, Clark DS. 1984. Effect of azone and propylene glycol on penetration of trifluorothymidine through skin and efficacy of different topical formulations against cutaneous herpes simplex virus infections in guinea pigs. Antimicrob Agents Chemother 26: 819-23 Riley RR, Duensing S, Brake T, Munger K, Lambert PF, Arbeit JM. 2003. Dissection of human papillomavirus E6 and E7 function in transgenic mouse models of cervical carcinogenesis.Cancer Res 63: 4862-71

  Currently, pre-cancerous lesions or early cervical cancer can be detected inexpensively, such as visual inspection of the cervix using acetic acid (vinegar) or Lugol's solution (iodine), or affordable to detect 14 types of high-risk HPV It can be detected by a new HPV-DNA test (Care HPV, Qiagen) that can be carried out at a reasonable price. However, there is a lack of affordable treatments for precancerous cervical dysplasia. All costly invasive treatments currently in use (cryotherapy, conical biopsy, and loop electrosurgical conical resection) are all typically medically trained in a specialized gynecological oncology department Both the person and the construction of the medical substrate. Such medical resources are not available to many high-risk people who do not have the economic and mobility means to access clinical services. Thus, although the limitations of screening in an environment with limited medical resources have been fully considered, alternative therapies are urgently needed worldwide for those who test positive. However, this requirement has not been met. The remaining challenge is to develop new treatments for precancerous cervical dysplastic lesions caused by high-risk HPV and to develop the lesions into in situ and / or invasive cervical cancer Is to prevent. Such treatment should be easy and inexpensive to implement at the local clinic, with the assistance of trained medical personnel, or by the patient himself. In addition, the above treatment is a single-site “screening, treatment, and prevention” public health care model that can be performed by even the most insidious people living in areas with severe temperature differences, limited space, and limited access to water and electricity. Should be effective as a single dose therapy that can be used in

  The present invention provides for the direct local delivery of one or more chemotherapeutic agents that treat cervical dysplasia to prevent cervical dysplasia from progressing to carcinoma in situ and / or invasive cervical cancer. As a non-invasive delivery system, a new copolymer and a new drug penetration enhancer are provided. Advantageously, the copolymer / accelerator according to the present invention is heat sensitive, mucoadhesive or skin adhesive, normal skin or skin lesions (transdermal delivery), or mucosa (transmucosal). Facilitates the penetration of large and small compounds with therapeutic activity, such as proteins larger than 52 amino acids through delivery. Such properties facilitate the delivery of a stable active compound contained in the copolymer / accelerator. The copolymer / promoter is a solid phase at room temperature to facilitate insertion into the cervical dysplasia area and local administration, and immediately dissolves at physiological temperature to adhere to the mucosa. Furthermore, it has the property of promoting the penetration of chemotherapeutic drugs throughout the squamous dysplasia. In a preferred embodiment, the copolymer / promoter is poloxamer 188 (P188), propylene glycol (PG), and laurocapram (LP) that can be customized and optimized to deliver one or more therapeutic agents or compounds. ).

  The present invention also provides a variety of therapeutic agents for treating neoplastic and non-neoplastic proliferative lesions of the skin or mucosal surface that are thermosensitive, skin-adhesive or mucoadhesive, and that promote penetration. It provides the use of copolymers / accelerators for local delivery. In embodiments, the copolymers / facilitators according to the present invention facilitate the delivery of therapeutic agents through normal skin or skin lesions or mucosal surfaces. The normal skin or skin lesion or mucosal surface includes the cervix, vagina, vulva, anus, rectum, eyes, ears, oral cavity, larynx, and head and neck for local treatment. It is not limited.

  In one embodiment, the present invention can be utilized to treat neoplastic and non-neoplastic proliferative diseases. Such proliferative diseases include premalignant low-grade cervical dysplasia and high-grade dysplasia (CIN3), including cervical intraepithelial neoplasia (CIN) 1 or 2 (CIN1, CIN2) HPV-related cervical lesions, vulvar cancer, vaginal cancer or vaginal tumor, endometrial cancer, laryngeal cancer, head and neck cancer, skin cancer, soft fibroma, general HPV-related hands, legs, limb warts As well as, but not limited to, psoriasis.

  In one embodiment, instead of subcutaneous injection using a needle or transvenous delivery using an indwelling catheter, transdermal delivery systems of large and small therapeutic agents and biomolecules to the subcutaneous tissue or submucosa, or systemic circulation or The present invention may be used as a transmucosal delivery system (or device).

* Shows thermal profile of Br1-containing P188 / PG / LP copolymer / accelerator. The differential scanning calorimetry curve of FIG. 1 contains 1 mM * Br1, 38 amino acid cytotoxic peptide, propylene glycol (PG) supplemented with 0.4 M laurocapram (LP) and poloxamer 188 (P188) in various ratios. The thermal properties of the drug delivery agent. A photomicrograph of mouse cervical squamous epithelium (10 times the original size) is shown. FIG. 2A shows H & E staining of the squamous epithelium covering the cervix and its left and right branches. Here, the squamous / columnar epithelial junction is not shown. FIG. 2B is a fluorescence micrograph of serial sections showing that the P188 / PG-containing drug delivery agent is dissolved and attached to the cervical mucosal surface at the left and right uterine horns. FIG. 2B ′ is an enlargement of the boxed area of panel B, not a drug delivery agent containing no LP (right cervix), but a drug delivery agent containing P188, PG, and LP (left In the cervical canal) at 6 mm after the insertion of the peptide-containing drug delivery agent at the thickness indicated by the arrow. Deep staining of mucoadhesive FITC conjugate * Br1 was observed on the mucosal surface of the cervix and left and right uterine horns. Deep staining showed that FITC junction * Br1 dissolved and adhered to the mucosal surface of the cervical dysplasia area extending to the cervix, cervix, and left and right uterine horns.

(Explanation of sequence listing)
SEQ ID NO: 1 shows the amino acid sequence of TRIP-Br1 decoy peptide (* Br1).
SEQ ID NO: 2 shows the amino acid sequence of TRIP-Br2 decoy peptide (* Br2).

  The present invention provides copolymers / accelerators for the topical transdermal or topical transmucosal delivery of therapeutic agents. Advantageously, the copolymer / accelerator according to the present invention is thermosensitive, mucoadhesive or skin-adhesive and promotes penetration of the therapeutic agent through the skin or mucosal surface. The present invention also provides the therapeutic use of copolymers / promoters for the local treatment of cancer, including cervical cancer.

  In a preferred embodiment, the delivery system comprises a copolymer of poloxamer 188 (P188) and propylene glycol, a penetration enhancing laurocapram, and optionally one or more therapeutic agents. In a preferred embodiment, a delivery agent according to the invention is used to produce large cytotoxic peptides such as TRIP-Br1 decoy peptides (* Br1) and / or TRIP-Br2 decoy peptides (* Br2) containing TRIP-Br1 decoy peptides (* Br2). Can be delivered locally. TRIP-Br decoy peptides are aimed at disrupting the TRIP-Br integration function in the E2F responsive promoter, which induces a new mechanism of cell death in proliferating cells. (Refer nonpatent literature 8-10.). Thus, local delivery of the TRIP-BR decoy peptide according to the present invention not only provides local treatment in precancerous cervical dysplasia, but also precancerous cervical dysplasia. Can prevent progression to cancer.

  As shown in the examples, the copolymer / accelerator according to the present invention is heat sensitive. That is, it is solid at room temperature to facilitate insertion into the cervical dysplasia area and dissolves immediately at physiological temperatures. Furthermore, the copolymer / promoter adheres to the peptide in the cervical mucosa and promotes peptide penetration.

  In one embodiment, using the present invention, cytologically as cervical intraepithelial neoplasia 1 or 2 (CIN1, CIN2) to prevent progression to carcinoma in situ (CIS) and invasive cervical cancer Local treatment of low-grade cervical dysplasia or high-grade dysplasia (CIN3) associated with human HPV as defined can be performed (see Non-Patent Document 11).

  Advantageously, the copolymer / accelerator according to the present invention allows non-invasive local delivery of therapeutic agents through the skin and mucosal surfaces. Furthermore, the copolymer / accelerator utilizes safe and inexpensive ingredients, is easy to administer, and is suitable for use in various clinical environments. The copolymer / accelerator is a medical worker or patient even in an environment where the temperature is extremely low or high, the humidity is high, the light is low, the place is limited, and sufficient supply of electricity and water cannot be obtained. It can be administered easily by itself.

(Copolymer / accelerator for local delivery of therapeutic agents)
In one aspect of the invention, a copolymer / accelerator for local delivery of a therapeutic agent is provided. Advantageously, the copolymer / accelerator according to the present invention is thermosensitive, mucoadhesive or skin-adhesive and promotes penetration of the therapeutic agent through full thickness skin or mucosa.

In one embodiment, the new topical delivery agent comprises a thermosensitive polymer, a mucoadhesive or skin adhesive polymer, a penetration enhancer, and optionally one or more therapeutic agents.

  In one embodiment, the copolymer / accelerator is a copolymer of one or more thermosensitive polymers, one or more mucoadhesive or skin-adhesive polymers, and one or more penetration enhancers. including. In a preferred embodiment, the copolymer / accelerator comprises a copolymer of poloxamer 188 and propylene glycol, a penetration enhancer laurocapram, and optionally one or more therapeutic agents.

  In one embodiment, the copolymer / accelerator comprises one or more polymeric materials. The one or more polymeric materials include, but are not limited to, poloxamers and poloxamines. Useful poloxamers according to the present invention include poloxamers 188, 407, 101, 105, 108, 122, 123, 124, 181, 182, 183, 184, 185, 212, 215, 217, 231, 234, 235, 237, 238, 282, 284, 288, 331, 333, 334, 335, 338, 401, 402, and 403. Useful poloxamines according to the present invention include poloxamines 304, 504, 701, 702, 704, 707, 901, 904, 908, 1101, 1102, 1104, 1301, 1302, 1304, 1307, 1501, 1502, 1504, and 1508. Including, but not limited to.

  In embodiments, the copolymer / accelerator comprises one or more polymeric materials, polyvinyl acetate, cellulose and derivatives thereof (carboxymethylcellulose, cellulose acetate, cellulose acetate propionate, ethylcellulose, hydroxypropylmethylcellulose, hydroxy Alkylmethylcelluloses and alkylcelluloses), cross-linked dextrans, polyethylene glycol, diethylaminoethyl dextran, poly (cyanoacrylates), copolymers of PEG and PLA, poly (lactic acid-glycolic acid copolymers), poly (Orthoesters), as well as hydrogels. The one or more polymeric materials include, but are not limited to, polylactic acid and copolymers. Preferably, the one or more polymeric materials are biodegradable materials that are pharmaceutically acceptable and have mucoadhesive or skin adhesion and / or allow penetration of the therapeutic agent through the skin and / or mucosal surface. Facilitate.

  In one embodiment, the copolymer / accelerator further comprises one or more mucoadhesive or skin adhesive materials. In one embodiment, the one or more mucoadhesive or skin-adhesive substances promote adhesion of the copolymer / promoter to the skin or mucosa, eg, cervical epithelium. Preferably, the one or more mucoadhesive or skin adhesive substances also promote penetration of the therapeutic agent through the skin and / or mucosal surface.

  Useful mucoadhesive or skin adhesive materials according to the present invention include polyols such as propylene glycol, dipropylene glycol, polyethylene glycol, glycerin, and butylene glycol, and glycerol esters such as oleates of propylene glycol. Including but not limited to glycol derivatives as well as nonionic glycol ether derivatives such as ethoxydiglycol.

  Useful mucoadhesive or skin-adhesive substances according to the present invention include polymers such as polyethylene glycol capryl / caprin glycerides, vinyl polymers (eg, polyhydroxyethyl acrylate, polyhydroxyethyl methacrylate, polyvinyl alcohol, and polyvinyl pyrrolidone), Cellulose derivatives such as methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and carboxymethylcellulose, and polysaccharides such as alginic acid and sodium alginate may also be included.

  In one embodiment, the topical delivery agent further comprises one or more penetration enhancers. Useful penetration enhancers according to the present invention include laurocapram, diethylene glycol, monoethyl ether, n-decylmethyl sulfoxide, dimethyl sulfoxide, dimethylacetamide dimethylformamide, sucrose monooleate, amides and other nitrogen compounds such as urea, 2-pyrrolidone, 1-methyl-2-pyrrolidone, ethanolamine, diethanolamine, and triethanolamine), organic acids (eg, citric acid and succinic acid), N-methyl-2-pyrrolidine, borage oil, tetrahydropiperine (THP) Alcohols (eg, methanol, ethanol, propanol, octanol, benzyl alcohol, myristyl alcohol, cetyl alcohol, and stearyl alcohol), fatty acids (eg, Ynoic acid), fatty acid esters (e.g., isopropyl myristate, isopropyl palmitate), polyols (e.g., propylene glycol, polyethylene glycol, and glycerol), polyethylene glycol monolaurate, and lecithin, but not limited to.

  In one embodiment, the penetration enhancer is a specific mucoadhesive or skin-adhesive substance that acts as a penetration enhancer when used in combination with propylene glycol, but acts as a penetration retarder when used in combination with polyethylene glycol. In combination, penetration can be accelerated or even delayed. Copolymer / retarding agents may be used to prevent harmful compounds from penetrating through the skin or mucosal surface. Such harmful compounds include, but are not limited to, toxins released during environmental or catastrophic events. In this embodiment, the copolymer / retarder is not only a chemical agent, biological agent, radioactive material, nuclear material, but also a kind of personal defense or medical against infectious agents, epidemic influenza, and other emerging infectious diseases. It may play a role as a countermeasure (MCM).

  Preferably, the copolymer / accelerator is solid or semi-solid at room temperature and dissolves at a temperature slightly below the physiological temperature. Generally, room temperature is less than 30 ° C, less than 28 ° C, less than 25 ° C, less than 23 ° C, less than 20 ° C, or less than 18 ° C.

  In embodiments, the copolymer / accelerator is dissolved in a temperature range of about 30 ° C to 42 ° C, 32 ° C to 40 ° C, 33 ° C to 40 ° C, 35 ° C to 38 ° C, or 34 ° C to 37 ° C. Or begin to dissolve. In embodiments, the copolymer / accelerator will or will begin to dissolve at temperatures above 30 ° C, 31 ° C, 32 ° C, 33 ° C, 34 ° C, 35 ° C, 36 ° C, or 37 ° C. In embodiments, the copolymer / accelerator is 45 ° C, 44 ° C, 43 ° C, 42 ° C, 41 ° C, 40 ° C, 39 ° C, 38 ° C, 37 ° C, 36 ° C, 35 ° C, or 34 ° C. It melts at a temperature below or starts to melt.

  The desired thermal properties of the copolymer / accelerator are the relative proportions (eg, weight percent or molar amount) of the various components including thermosensitive polymeric materials, mucoadhesive materials, penetration enhancers, and / or therapeutic agents. This ratio can be realized by adjusting the ratio.

  In embodiments, the copolymer / accelerator has a weight percent of about 20% to about 95%, about 25% to about 90%, about 30% to about 85%, about 35% to about 80%, about 40% to about 70%, about 50% to about 90%, about 50% to about 85%, about 60% to about 80%, about 30% to about 40%, about 30% to about 50%, about 70% From about 90%, from about 70% to about 85%, or from about 70% to about 80%.

  In embodiments, the copolymer / accelerator has a weight percent of about 5% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 20%, about 5% to about 30%, about 5% To about 20%, about 5% to about 15%, or about 15% to about 30%.

  In embodiments, the copolymer / accelerator or the copolymer / retarder has a concentration of about 0.1 M to about 1 M, about 0.2 M to about 0.9 M, about 0.3 M to about 0.00. Permeation enhancers or penetration retarders that are 8M, about 0.4M to about 0.7M, or about 0.2M to about 0.5M, respectively.

  In embodiments, the copolymer / accelerator or the copolymer / retarder has a concentration of 0.05M, 0.1M, 0.15M, 0.2M, 0.3M, 0.4M,. Includes penetration enhancers or penetration retarders greater than 5M, 0.6M, 0.7M, 0.8M, 0.9M, 1.0M, 1.5M, 2M, 2.5M, or 3M, respectively.

  In embodiments, the copolymer / accelerator or the copolymer / retarder is at a concentration of 7M, 6M, 5M, 4.5M, 4M, 3.5M, 3M, 2.5M, 2M, 1.M. 5M, 1M, 0.9M, 0.8M, 0.7M, 0.6M or less than 0.5M penetration enhancer or penetration retarder, respectively.

  In certain embodiments, the copolymer / accelerator comprises poloxamer 188 and propylene glycol at about 100: 0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30. : 70, 20:80, 10:90, or 0: 100 (w / w). In a preferred embodiment, the copolymer accelerator comprises poloxamer 188 and propylene glycol at a ratio of about 70:30 (core body temperature 37 ° C.) or 65/35 (skin temperature 34 ° C.) (w / w).

  In embodiments, the copolymer / accelerator has a concentration of about 0.1 M to about 1 M, about 0.2 M to about 0.9 M, about 0.3 M to about 0.8 M, about 0.4 M to about Laurocouplers that are 0.7M, or about 0.2M to about 0.5M. In a preferred embodiment, the copolymer / accelerator comprises about 0.4M laurocapram.

  The copolymers / accelerators can be used to locally deliver large and small therapeutic agents not previously realized with penetration enhancers. Penetration enhancers include peptides and proteins larger than 52 amino acids, nucleic acids, unique physicochemical structures and / or properties that were not considered suitable for transdermal or transmucosal delivery Chemotherapeutic agents, anticancer or antitumor agents, antibiotics, antibacterial agents, antiviral agents, antifungal agents, antimicrobial agents, anti-neoplastic agents, immunomodulators, anti-inflammatory agents, cytokines and chemokines (e.g. Leukins), substances suitable for the treatment of diabetes, such as insulin preparations with or without secretagogues and / or thiazolidinediones, substances suitable for acute and / or chronic anticoagulation (eg low molecular weight heparin), and currently Includes vaccine antigens administered by subcutaneous or intramuscular injection or intravenous delivery via an indwelling catheter. It is but is not limited thereto. In a preferred embodiment, the copolymer / facilitator transdermally delivers large therapeutic biomolecules such as insulin, antithrombotic agents, and vaccine antigens for local delivery of therapeutic agents to treat cervical dysplasia. Useful for delivery or transmucosal delivery.

  Conversely, the copolymer / retarder can be used to prevent penetration of harmful compounds through the skin or mucosal surface. Such harmful compounds include, but are not limited to, toxins released during environmental or catastrophic events. In this embodiment, the copolymer / retarder is not only a chemical agent, biological agent, radioactive material, nuclear material, but also a kind of personal defense or medical against infectious agents, epidemic influenza, and other emerging infectious diseases. It may play a role as a countermeasure (MCM).

  Examples of decoy peptides used in the formulations of the present disclosure include TRIP-Br1 decoy peptide (* Br1) (ATGCLDGDGLEGLFEDID) (SEQ ID NO: 1) described in US Pat. No. 7,223,733 (see claims 1-5) and A TRIP-Br decoy peptide such as TRIP-Br2 decoy peptide (* Br2) (TGLTDLTLDDDILFADID) (SEQ ID NO: 2) can be mentioned, but is not limited thereto.

  Examples of chemotherapeutic and anticancer / antitumor agents used in the formulations of the present disclosure include 5-fluorouracil, chlorambucil, aminolevulinic acid, altretamine, ambomycin, vincristine, butionine sulfoximine, asparaginase, bleomycin, insulin, trimeme Trexate, adriamycin, taxotere, carboplatin, cisplatin, carmastine, cladribine, 5-ethynyluracil, 9-dihydrotaxol, mitomycin, abiraterone, acivicin, teniposide, aclarubicin, acodazole hydrochloride, canarypox IL-2, acronin, thioguanine, acylfulvene , Adecipenol, adzelesin, aldesleukin, thiotepa, ambermustine, busulfan, amethadrone acetate, Midox, amrubicin, mercaptopurine, cyclophosphamide, cytarabine, paclitaxel, pentostatin, dacarbazine, dactinomycin, daunorubicin, camptothecin derivatives, doxorubicin, etoposide, fludarabine phosphate, hydroxyurea, BRC / ABL antagonist, brefralate, brequinal Sodium, bropirimine, budotitanium, amifostine, actinomycin, calcipotriol, calphostin C, carsterone, calacemide, carbetimer, floxuridine, idarubicin, ifosfamide, lomustine, mechlorethamine, melphalan, methotrexate, mitoxantrone, pricamycin, procarbazine, Streptozocin and vinblastine include but are not limited to Not.

  In one embodiment, the vaccine antigen may be, for example, a tumor antigen or an antigen of a pathogen such as a virus, bacteria, fungus, and parasite. Thus, in some embodiments, the antigen is, for example, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus (HSV), human immunodeficiency virus. (HIV), human papillomavirus (HPV), cytomegalovirus (CMV), influenza viruses (eg, influenza A virus), and viruses such as rabies virus, but are not limited thereto. In other embodiments, the antigen is, for example, Vibrio cholerae, Diphtheria, Tetanus, Streptococcus (eg, Streptococcus group A and B), Streptococcus pneumoniae (eg, 90 or more serotypes, 88% of them) Causes invasive disease and is included in 23-valent polysaccharide vaccines), pertussis, meningococci (eg, meningitis A, B, C, W, and Y), Neisseria gonorrhoeae, Helicobacter pylori And from bacteria such as Haemophilus influenzae (eg, Haemophilus influenzae type B). In yet another embodiment, the antigen containing the polypeptide is obtained from a parasite such as, for example, a malaria parasite. Also included are antigens used to immunize against infant diseases such as polio, measles, mumps, and rubella.

  In yet another embodiment, substances suitable for the treatment of diabetes (types 1 and 2), as well as thrombosis and pro-thrombotic conditions (eg deep vein thrombosis, pulmonary embolism, chronic atrial fibrillation, prosthetic heart valve) And substances containing substances suitable for the prevention and treatment of sickle cells). Examples of such substances suitable for the treatment of diabetes include insulin and insulin analogs such as lispro (Humalog), humanine (Isophene and Regular), nobolog (Aspart), levemil (Detemil), and Lantus (Glargin) )) As well as other small molecules such as, but not limited to, metformin, rosiglitazone, pioglitazone, and combinations containing such molecules (eg, metformin and rosiglitazone, rosiglitazone and glimepiride) . Examples of other compounds that can be incorporated into the formulations of the present disclosure include biguanides such as metformin (glucophage), thiazolidinediones such as rosiglitazone and pioglitazone (TZDs), tolbutamide (orinase), acetohexamide (dimerol) , Sulfonylureas such as tolazamide (trinase), chlorpropamide (diabines), glipizide (glucotrol), glyburide (diabeta, micronase, and glycanase), glimepiride (amaril), or gliclazide (diamicron), meglitinides ( For example, non-sulfonylurea secretagogues such as repaglinide (plangin) and nateglinide (starix)), and miglitol (glycet) or acarbose (precourse, Fences), and the α- glucosidase inhibitors such as. Examples of such substances for the prevention or treatment of acute and / or chronic prethrombotic or thrombotic conditions include: ardeparin (normifuro), bemiparin (hiball, ybor, dibor, and buddyket), sertoparin (sandparin), Unfractionated heparin and low molecular weight heparins such as dalteparin (fragmin), enoxaparin (labnox, clexane), nadroparin (flaxiparin), parnaparin (fluxam), leviparin (crivaline), and tinzaparin (inohep, loparin), fondaparinux ( Alixtra) and Idraparinux sodium (SANORG 34006, SR34006), rivaroxaban (BAY59-7939, Exarelto), and apixaban (Ericus) Direct thrombin inhibitors such as lepirdin (refludane), bivalirudin (angiomax or angiox), argatroban (acova, arganova, argatra, and novastan), and dabigatran (pradaxa, pradax, and plazaxa) And vitamin K antagonists such as, but not limited to, warfarin (coumadin), acenocoumarol (syntron, synthrom), and fenprocomon (Markumar, Marcmar, and faritron). In embodiments, the copolymer / enhancer is at a concentration of about 0.1 mM to about 3 mM, about 0.1 mM to about 2 mM, about 1 mM to about 1.5 mM, about 0.5 mM to about 2 mM, or about A therapeutic agent that is 0.5 mM to about 1.5 mM is included. The amount of therapeutic agent incorporated into the copolymer / promoter of the present disclosure is such that the therapeutic agent is delivered in an amount that provides a therapeutic effect on the subject (eg, based on age, bioavailability of the therapeutic agent, etc.). As such, it can be determined by one skilled in the art.

(Treatment of cancer by local delivery of therapeutic agents)
In another aspect of the invention, a method is provided that facilitates delivery of a therapeutic agent across the skin and / or mucosal surface. Preferably, the present invention facilitates non-invasive local delivery of a wide variety of therapeutic agents (eg, peptides, proteins, chemicals, and nucleic acids) of various types and compositions with various physicochemical properties. The therapeutic agents include, as described above, cytotoxic decoy peptides, other chemotherapeutic agents, and anti-tumor / anti-cancer agents, therapeutic agents for diabetes, prophylactic and therapeutic agents for thrombotic and prothrombotic conditions, and various Antigens for inducing a protective immune response against vaccine antigens are included, but are not limited to these.

  In one embodiment, the method comprises administering a copolymer / accelerator according to the present invention to the skin or mucosal surface of a subject. In certain embodiments, the method includes applying a copolymer / promoter containing a copolymer of poloxamer 188 and propylene glycol, laurocapram, and optionally one or more therapeutic agents to the skin or mucosal surface of a subject. Administration.

  As used herein, the term “subject” refers to an organism, including mammals such as primates, that can be treated with a formulation according to the present invention. Examples of mammalian species that can benefit from the treatment methods of the present disclosure include apes, chimpanzees, orangutans, humans and monkeys, and dogs, cats, horses, cows, pigs, sheep, goats, chickens, mice But not limited to, domestic animals such as rats, guinea pigs, and hamsters.

  In embodiments, the copolymer / accelerator according to the present invention is administered to the skin or mucosal surface. Such skin or mucosal surfaces include, but are not limited to, the cervix, vagina, anus, rectum, eyes, ears, nose, chest, vulva, larynx, and head and neck. In one embodiment, the copolymers / promoters of the present invention are delivered locally to the cervical vagina and cervical dysplastic lesions near the cervix in females by intravaginal administration, a mucosal delivery.

  In one embodiment, the present invention allows local delivery of a therapeutic agent through the keratinized top layer (stratum corneum) and / or mucosa of the skin. In other embodiments, the present invention allows for local delivery of therapeutic agents through the non-keratinized surface of the skin and / or mucosa. In one embodiment, the present invention allows for local delivery of a therapeutic agent to a multilayer of cervical squamous cells or local delivery of a therapeutic agent through a multilayer of cervical squamous cells. In one embodiment, the present invention allows for local delivery of therapeutic agents to the basal keratinocytes and / or mucosa of the skin.

  In certain embodiments, the present invention can be used to deliver a therapeutic agent that penetrates to the keratinized surface of the mouse cervical dysplasia area. In certain embodiments, the invention is used to deliver a therapeutic agent that penetrates through a multilayer of squamous cells to a non-keratinized human cervical dysplasia region (T-region), causing HPV virus integration to occur. Can reach the basal keratinocytes.

  As illustrated in the Examples, the copolymers / accelerators of the present invention can deliver moderately sized peptides through the keratinized top layer of the mouse cervical T-region. The mouse cervical dysplasia region is different from the human cervical dysplasia region. Although the mouse cervix is completely internalized, humans have a cervical vagina on the vaginal surface. In addition, the mouse cervix has a keratinized apical layer in the cervical T-region that begins at the vaginal-cervical junction and ends at the squamous-columnar junction. The cervical T-region is where most cervical cancer occurs. The keratinized cervical epithelium of mice corresponds to keratinized skin. As illustrated in the Examples, the copolymer / promoter allows local delivery and penetration of 38 amino acid cytotoxic peptides through the keratinized cervical squamous epithelium of mice.

  In embodiments, the present invention can be used to locally deliver therapeutic agents through the stratum corneum rich in skin keratin to treat cutaneous neoplastic and non-neoplastic proliferative disorders. Examples of the above skin neoplastic and non-neoplastic proliferative diseases include precancerous and cancerous conditions related to HPV affecting the vulva, vagina, anus, larynx, head and neck, melanoma, basal cell carcinoma Nasopharyngeal cancer associated with Epstein-Barr virus infection, as well as psoriasis.

  In one embodiment, the present invention can be used to locally deliver anti-cancer / anti-tumor and chemotherapeutic agents for treating tumors or cancer. The anticancer / antitumor agents include premalignant low-grade cervical dysplasia, high-grade dysplasia (CIN3), classified as cervical intraepithelial neoplasia 1 or 2 (CIN1, CIN2), Intraepithelial carcinoma (CIS), and human HPV-related cervical cancer and its precursor lesions such as locally invasive cervical cancer or metastatic cervical cancer (see Non-Patent Document 11), prostate cancer, ovarian cancer, vulvar cancer, vaginal cancer, Or include, but are not limited to, vaginal tumors, endometrial cancer, laryngeal cancer, bladder cancer, nasopharyngeal cancer, skin cancer, and head and neck cancer.

  In embodiments, the present invention can be used to non-invasively locally deliver a therapeutic agent for a disease or condition. Such diseases or conditions include proliferative skin lesions such as genital warts, psoriasis and keloids.

  In embodiments, the book is a non-invasive local transdermal delivery system or non-invasive local transmucosal delivery system (or device) that is applied to normal skin or mucosal surfaces to eliminate the need for subcutaneous injection of the therapeutic agent. The invention can be used.

(Formulations and formulations for topical administration)
According to the present invention there is provided a therapeutic or drug formulation comprising a copolymer / accelerator in a form that can be combined with a pharmaceutically acceptable carrier. In a preferred embodiment, the therapeutic or drug formulation is a solid or semi-solid at room temperature and dissolves at a temperature slightly below the desired physiological temperature.

  The term “carrier” refers to a diluent, adjuvant, additive, or vehicle with which the formulation can be administered. Such a drug carrier can be a sterile liquid such as water or oil. The sterilizing liquid includes a sterilizing liquid such as petroleum oil such as mineral oil, food oil such as peanut oil, soybean oil, and sesame oil, animal oil, or synthetic oil.

  Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skim milk powder, glycerol, propylene Glycol, water, ethanol and the like are included. The therapeutic agent may contain a small amount of a wetting agent, an emulsifying agent or a pH buffering agent as required. The formulations can take the form of creams, foams, patches, lotions, infusions, sprays, gels, oils, aerosols, powders, ointments, solutions, suspensions, emulsions, and the like. The formulations can be formulated with conventional binders and carriers such as triglycerides. Examples of suitable drug carriers are E.I. W. Martin's "Remington's Pharmaceutical Sciences". The formulation will contain an appropriate amount of the carrier along with an effective amount of the therapeutic agent to provide a form for proper administration to the patient. The dosage form of the formulation should be adapted to the method of administration.

  According to the present invention, it is possible to provide a component that is stable even when administered to a subject compared to before modification, that is, modified so that the effect lasts for a long time when administered. Such modifications are well known to those skilled in the art, for example, microencapsulation.

  The amount of the therapeutic agent or drug preparation of the present invention effective for treating a specific disease, condition or disorder depends on the route of administration and the severity of the disease, condition or disorder, and can be determined by the practitioner and each patient's situation. Therefore, it should be decided.

  Furthermore, the present invention provides kits containing therapeutic agents such as lyophilized cytotoxic decoy peptides, vehicles, and / or copolymers / promoters. Preferably, the formulations of the present invention are stable over a wide range of temperatures below the desired dissolution temperature. In one embodiment, the active therapeutic agent can be reconstituted by mixing pre-measured amounts of each component immediately prior to use.

<Example>
The following are examples showing procedures for carrying out the present invention. This example should not be construed as limiting. Unless otherwise noted, all percentages are by weight and all solvent mixing ratios are by volume.

<Example 1: Heat-sensitive mucoadhesive penetration enhancer>
To produce copolymers / accelerators with the desired heat sensitivity, various proportions (100/0) of poloxamer 188 (P188) and propylene glycol (PG) meeting US Pharmacopeia (USP) grade standards. 80/20, 70/30, 50/50, and 0/100) and heated to 60 ° C. The resulting copolymer / accelerator is sucked into a preheated (60 ° C.) silastic tube (1/8 inch inner diameter, Fisher, Pittsburgh, PA), solidified at room temperature, and compressed air from the tube Extruded (see Non-Patent Document 12). A 3 mm sample was cut from the obtained “rope” solid using a scalpel.

  The thermal profile of the P188 / PG copolymer preparation was determined using a differential scanning calorimeter DSC6200 / Exstar 6000 (Seiko Instruments Inc.). In order to obtain a copolymer formulation having the desired melting point (30-37 ° C.) in the presence of 1 mM of various decoy peptides, DSC 6200 was heated at 10 ° C./min, cooled at 50 ° C./min, Three cycles were programmed to take 2 minutes equilibration time between heating / cooling cycles. The measurement was performed in the temperature range of −25 to 65 ° C.

  The DSC curve did not show a double peak, indicating that the P188 / PG copolymer formulation was a homogeneous phase. A copolymer with a P188 / PG ratio of 80/20 exhibited the desired thermal properties. That is, it was solid at room temperature and immediately dissolved at a temperature slightly below the physiological temperature of 37 ° C.

  A P188 / PG (80/20 ratio) copolymer was then used to deliver the FITC labeled 38 amino acid cytotoxic decoy peptide. As determined by direct visualization using fluorescence microscopy, the P188 / PG copolymer (80/20 ratio) alone was unable to penetrate the cornified apical layer of the mouse cervix (data not shown) ).

  Laurocapram is a non-toxic skin penetration enhancer (see Non-Patent Documents 13 and 14). Laurocapram is a type of “penetration regulator” (see Non-Patent Documents 15 and 16) that can promote or retard penetration in the human stratum corneum depending on the medium. DSC and spectral analysis revealed that penetration modifiers (eg, laurocapram) disrupt and fluidize the stratum corneum lipid bilayer (see Non-Patent Document 17). Laurocapram has been widely used as a safe penetration enhancer since 1984, and contains propylene glycol, a mucoadhesive and skin adhesive substance (see Non-Patent Documents 17 and 18).

  0.4M Laurocapram (see Non-Patent Document 17) was added to the P188 / PG formulation. The DSC curve (FIG. 1) shows that a formulation consisting of 1 mM cytotoxic decoy peptide * Br1, P188 / PG (70/30 ratio) copolymer, and 0.4 M laurocapram is used for delivery of cytotoxic decoy peptide. (The delivery of Br2 is not shown). As shown below, the new formulation can promote the penetration of cytotoxic decoy peptides through the keratinized surface of the mouse cervical mucosa, which resembles the stratum corneum of the skin.

  The extent of the entire mouse cervical T-region is from the cervical-vaginal junction to the end of the cervical T-region and is approximately one third of the distance to one of the left and right uterine horns. It is. For local delivery of decoy peptides throughout the mouse cervical T-region, a rod-shaped copolymer / promoter containing FITC-labeled solid decoy peptides expresses HPV16E6 oncoprotein that cooperates with 17β-estradiol A human child in the “untreated” cervix delivered manually to one of the left and right uterine horns and the cervix in a 7 month old female K14E6 homozygous transgenic mouse and served as an internal control In situ low grade dysplasia (CIN1, 2) was induced which almost reproduces the gradual progression of cervical cancer.

  After the animal was anesthetized, a “microscope” containing a typical P-100 pipette tip with the tip resected along its length was inserted into the vagina to expose the cervical opening . A 3/4 inch long 24 gauge catheter (Terumo Surfflash, 0.47 mm ID) with the trocar removed was attached to a compatible P-10 micropipette. Until the length of the entire cervical T-region and the length leading to the uterine horn through the squamous / cylindrical epithelial junction are combined, the catheter is placed with a liquid peptide-containing copolymer / promoter. Satisfied.

  After the copolymer / accelerator was solidified into a rod shape at room temperature, the catheter tip was inserted into an opening of the cervix, and the copolymer / accelerator was extruded into the cervix using a trocar. Meanwhile, at the end of the procedure, the catheter tip was withdrawn so that the copolymer / promoter protruded 2 mm outside the cervix. The copolymer / accelerator was observed to consistently transition from a rod-like solid to a gel-like formulation without leaking from the cervical opening.

  The animals were euthanized 6 hours after peptide delivery. The entire reproductive organ was surgically removed and fixed with formalin to prepare a tissue section (see Non-Patent Document 19).

  Direct visualization of peptide delivery was performed by fluorescence microscopy using 1 mM FITC-conjugated * Br1. Fluorescence microscopy showed that both formulations containing P188 and PG had mucoadhesive properties on the keratinized top surface.

  FIG. 2 is a direct comparison of the penetration characteristics of a laurocapram-containing formulation and the laurocapram-free formulation in the reproductive tract of one mouse. Under ultrasound guidance, a rod-shaped P188 / PG (80/20 ratio) formulation (without laurocapram) was introduced at a fairly high position in the right uterine horn and rod-shaped P188 / PG with 0.4 M laurocapram. (70/30 ratio) The formulation was introduced into the left uterine horn.

  In particular, permeation through the keratin apical layer of the cervical squamous epithelium was observed only in the left uterine horn introduced with a rod-shaped P188 / PG (70/30 ratio) preparation containing 0.4 M laurocapram (Fig. 2B, 2B ′). Comparing fluorescence microscopy imaging of unstained sections (FIG. 2B) with optical microscopy of serial H & E stained sections (FIG. 2A), rod-shaped P188 / PG (70/30) containing laurocapram through a multilayer of cervical squamous epithelial cells. It is clear that the formulation penetrated.

  All patents, patent applications, provisional patent applications, and publications referred to or cited herein are hereby incorporated by reference in their entirety, including all figures and tables, to the extent that they do not conflict with the obvious teachings of this specification. Incorporated as.

  It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or alterations that take into consideration are suggested to those skilled in the art and are within the spirit and scope of the present application. It will be understood.

Claims (21)

A topical delivery agent for local delivery of a therapeutic agent through the skin or mucosal surface,
Poloxamer 188,
Propylene glycol,
Laurocapram,
a) Decoy peptides, antibiotics, antibacterial agents, antiviral agents, antifungal agents, antimicrobial agents, immunomodulators, anti-inflammatory agents, cytokines or chemokines b) Insulin, insulin analogues, biguanides, thiazolidinediones, sulfonylureas, C) metformin, rosiglitazone, pioglitazone, troglitazone, tolbutamide, acetohexamide, tolazamide, chlorpropamide, glipizide, glyburide, glimepiride, gliclazide, including non-sulfonylurea secretagogues, α-glucosidase inhibitors Repaglinide, nateglinide, miglitol, acarbose, or combinations thereof d) TRIP-Br1 decoy peptide (* Br1) (ATGCLLDGGLEGLFEDID) (SEQ ID NO: 1), or one or more decoy peptides selected from TRIP-Br2 decoy peptide (* Br2) (TGLTDLTLDDILFADID) (SEQ ID NO: 2) e) an antithrombotic agent,
f) 38 amino acid cytotoxic decoy peptide,
g) A topical delivery agent comprising a therapeutic agent selected from a) to g) of a vaccine antigen. The topical delivery agent of claim 1, comprising
The therapeutic agent is a 38 amino acid cytotoxic decoy peptide. The topical delivery agent of claim 1, comprising
The local delivery agent delivers the therapeutic agent noninvasively locally to the skin or mucosal surface of a subject. A topical delivery agent according to claim 3,
The topical delivery agent wherein the skin or mucosal surface is the skin or mucosal surface of the cervix, vagina, anus, rectum, eyes, ears, nose, chest, vulva, pharynx, head, or neck. The topical delivery agent of claim 1, comprising
The therapeutic agent is a local delivery agent selected from decoy peptides, antibiotics, antibacterial agents, antiviral agents, antifungal agents, antimicrobial agents, immunomodulators, anti-inflammatory agents, cytokines, or chemokines. The topical delivery agent of claim 1, comprising
The therapeutic agent is 1 selected from TRIP-Br1 decoy peptide (* Br1) (ATGCLDLDGLEGLFEDID) (SEQ ID NO: 1) or TRIP-Br2 decoy peptide (* Br2) (TGFLTDLTLDILFADID) (SEQ ID NO: 2) A topical delivery agent that is one or more decoy peptides. The topical delivery agent of claim 1, comprising
The therapeutic agent includes insulin, insulin analog, biguanide, thiazolidinedione, sulfonylurea, non-sulfonylurea secretagogue, α-glucosidase inhibitor, or a combination thereof. A topical delivery agent according to claim 7 comprising:
The insulin analogues are insulin Lisp Russia, insulin Isofe down, insulin Asupato, topical delivery agent is insulin detemir, or insulin Gurarugi down. The topical delivery agent of claim 1, comprising
The therapeutic agent is metformin, rosiglitazone, pioglitazone, troglitazone, tolbutamide, acetohexamide, tolazamide, chlorpropamide, glipizide, glyburide, glimepiride, gliclazide, repaglinide, nateglinide, miglitol, acarbose, or a combination thereof . A topical delivery agent according to any one of claims 7 to 9,
The local delivery agent is administered to a subject to treat diabetes. A topical delivery agent according to claim 10 comprising:
The subject is a mammalian laboratory animal model or a human topical delivery agent. The topical delivery agent of claim 1, comprising
The therapeutic agent is a vaccine antigen. A topical delivery agent according to claim 12, comprising:
The vaccine antigen is selected from the group consisting of a tumor antigen, a viral antigen, a bacterial antigen, a fungal antigen, a parasite antigen, and an allergen. A topical delivery agent according to claim 12, comprising:
The vaccine antigen includes hepatitis A virus (HAV) antigen, hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) antigen, herpes simplex virus (HSV) antigen, human immunodeficiency virus (HIV) antigen, human papilloma Virus (HPV) antigen, cytomegalovirus (CMV) antigen, influenza virus antigen, rabies virus antigen, cholera antigen, diphtheria antigen, tetanus antigen, streptococcal antigen, Streptococcus pneumoniae antigen, pertussis antigen, meningococcal antigen, Neisseria gonorrhoeae A topical delivery agent that is an antigen, Helicobacter pylori antigen, Haemophilus influenzae antigen, Polio antigen, Measles antigen, Mumps antigen, or Rubella antigen. 15. A topical delivery agent according to any one of claims 12 to 14, comprising
The local delivery agent is administered to induce an immune response in a subject. A topical delivery agent according to claim 15,
The subject is a mammalian laboratory animal model or a human topical delivery agent. The topical delivery agent of claim 1, comprising
The therapeutic agent is an antithrombotic agent administered to prevent or treat a thrombus formation-promoting state or a thrombotic state. The topical delivery agent of claim 17, comprising
The antithrombotic agent is selected from the group consisting of unfractionated heparin, low molecular weight heparin, factor Xa inhibitor, direct thrombin inhibitor, and vitamin K antagonist. The topical delivery agent of claim 17, comprising
The antithrombotic agent is
Arudepari down, Bemipari down, Serutopari down, Darutepari down, Enokisapari down, Nadoropari down, Parunapari down, Rebipari down or Chinzapari down or we unfractionated heparin or low molecular weight heparin is selected, Fonda Paris quinuclidine scan, idraparinux sodium U arm, factor Xa inhibitor selected Ribarokisaba down or Apikisaba down or al, Repiruji down, Bibariruji down, Arugatoroba down or Dabigatora direct thrombin inhibitors are down or we select or warfarin down,, Asenokumaro Le or Fenpurokumo, topical delivery agent is a vitamin K antagonist selected down or al. A topical delivery agent according to any one of claims 17 to 19, comprising
The local delivery agent is administered to a subject to prevent or treat a thrombus formation-promoting state or a thrombotic state. The topical delivery agent of claim 20, comprising:
The subject is a mammalian laboratory animal model or a human topical delivery agent.